The present invention provides methods useful in operating a mass spectrometer incorporating a linear ion trap to simultaneously confine ions of opposite polarity in the linear ion trap and to react said ions inside the linear ion trap by charge transfer reactions, like electron transfer dissociation (ETD), negative electron transfer dissociation (NETD) or charge reducing proton transfer reactions (PTR).
A mass spectrometer typically comprises an ion source, a mass analyzer and an ion detector. Analyte substances can be ionized with a variety of techniques, for example by electron impact ionization (EI), chemical ionization (CI), electrospray ionization (ESI) or matrix assisted laser desorption/ionization (MALDI). The analyte ions are guided from the ion source to the mass analyzer. Any mass analyzer separates ions according on their mass to charge ratio, m/z, where m is the mass of the ions and z is the number of elementary charges of the analyte ions, i.e. the number of excess protons or electrons. Whenever the “mass of the ions” is referred to below, it is normally to be understood as the charge-related mass m/z. The separation can be in time, e.g., in a time-of-flight analyzer, in space, e.g. in a magnetic sector analyzer, or in a frequency space, e.g. in an ion cyclotron resonance cell (ICR). The analyte ions can also be separated according to their stability in a radio frequency (RF) multipole ion trap (two-dimensional or three-dimensional quadrupole ion trap) or a quadrupole filter. The mass selectively separated analyte ions are detected by an ion detector providing electronic data to construct a mass spectrum (MS) of the analyte ions.
In so called tandem mass spectrometers, selected precursor ions (also called parent ions) are first isolated, and then fragmented into fragment ions (also called daughter ions). The measured mass spectra of fragment ions (MS/MS) are useful to determine structural components of the precursor ions, e.g. the sequence of the amino acids of a peptide. Second generation fragment spectra (also called granddaughter ions) can also be measured as fragment ion spectra of isolated and fragmented daughter ions.
In the mid 1990's, McLuckey and coworkers pioneered the characterization of reactions between ions of opposite polarities inside three dimensional quadrupole ion traps (McLuckey et al., Mass Spectrometry Reviews, 1998, vol. 17, p. 369-407: “Ion/Ion Chemistry Of High-Mass Multiply Charged Ions”). The observed ion-ion-reactions include reactions between multiply charged positive ions with singly charged negative ions and reactions between multiply charged negative ions with singly charged positive ions, e.g. by proton transfers and electron transfers.
Three-dimensional quadrupole ion traps (3D ion traps) comprise a ring electrode and two end cap electrodes. The ring electrode is usually supplied with a one-phase RF voltage while the end cap electrodes are basically grounded; other modes of operation are possible. In the interior of the 3D ion trap, a RF quadrupole field is generated which oscillates with the frequency of the RF voltage. The quadrupolar RF field tends to drive ions towards the center of the trap. The restoring force—i.e. the force pushing ions towards the center of the trap—in the 3D ion trap is usually described by a so-called pseudo-potential. The pseudo-potential is determined by temporally averaging the effects of the real electric RF field on the ions. The pseudo-potential increases uniformly and quadratic in all directions from the center of the trap and is effective for both polarities. That is, both positive and negative ions can be stored simultaneously in the 3D ion trap. Therefore, 3D ion traps are well suited for reactions between positive and negative ions.
However, 3D traps have the disadvantage that they are not readily interfaced with downstream ion optics or analyzers. That is, after ions have been injected into, and reacted in a 3D trap, they cannot be easily extracted as a low energy ion beam. Because the ions ejected from the 3D trap have a broad distribution of ion energies, it is difficult to capture, guide, or analyze these ions in downstream devices.
In contrast, two dimensional multipoles are readily interfaced with upstream and downstream devices. Two dimensional quadrupole ion traps (2D ion traps, linear ion traps) are typically designed as multipole rod systems, e.g. as quadrupole, hexapole or octopole rod systems having two, three or four pairs of pole rods arranged symmetrically about a central axis. An RF voltage is applied in a first phase to every second rod and in an opposite phase to the remaining rods for generating a radially repelling pseudo-potential inside the linear ion trap. Quadrupole rod systems exhibit a quadratic rise in the pseudo-potential with radial distance from the central axis. Under the influence of the radially confining RF field and via collisions with a damping gas, ions are accumulated as a thread-like cloud along the central axis. 2D ion traps have two ends along the central axis. Ions may be injected into and ejected out of the trap along the central axis from either end. In the axial direction of the linear ion trap, ions have traditionally been confined by DC potentials applied to the rods or other electrodes, such as apertured electrodes placed at the ends of the linear trap. In the elongated volume of the linear ion trap defined by the rods, the DC potentials generate electrostatic fields that axially confine either positive ions or negative ions, but cannot simultaneously confine both.
The basic principle for confining ions of both polarities inside a linear ion trap has been known for a long time. U.S. Pat. No. 5,572,035 A (Franzen) discloses that: “All types of cylindrical or conical ion guides [ . . . ] can be used as storage devices if the end openings are barred for the exit of ions by reflecting RF or DC potentials. With RF field reflection, ions of both polarities can be stored. With DC potentials, ion guides store ions of a single polarity only.” The cited '035 patent generally discloses that ions of both polarities are repelled in the axial direction at a pseudo-potential barrier formed by inhomogeneous RF fields at the ends of linear ion traps. Further embodiments of linear ion traps with pseudo-potential barriers are disclosed in following: U.S. Pat. No. 7,026,613 B2 (Syka: “Confining positive and negative ions with fast oscillating electric potentials”), U.S. Pat. No. 7,227,130 B2 (Hager: “Method for Providing Barrier Fields at the Entrance and Exit End of a Mass Spectrometer”), U.S. Pat. No. 7,288,761 B2 (Collings: “System and method for trapping ions”) and U.S. Pat. No. 7,557,344 B2 (Chernushevich: “Confining Ions with Fast-Oscillating Electric Fields”). All aforementioned linear ion traps confine ions of both polarities by pseudo-potential barriers at both ends of the linear ion trap. An further overview of RF devices used to study ion-ion reactions is provided in the review article by Y. Xia and S. A. McLuckey (Xia et al., Journal of the American Society for Mass Spectrometry, 2008, vol. 19, p. 173-189: “Evolution of Instrumentation for the Study of Gas-Phase Ion/Ion Chemistry via Mass Spectrometry”)
One difficulty with the prior art method of confining ions in a linear ion trap via pseudo-potential barriers at both ends of the trap is that the “height” of these axial pseudo-potential barriers is dependent on the mass of the ion. Thus, if a precursor ion is very massive and either the reagent or product ions are very light, then the pseudo-potential barriers may not be able to axially confine both types of ions simultaneously because the pseudo-potential is inversely proportional to the mass of the ions.
As an alternative to rod systems, linear ion traps can be designed as a set of electrodes arranged along an axis as a stack of ring electrodes. Such prior art devices include RF ion funnels or RF ion tunnels, or a stack of apertured electrodes having opposing hyperbolic indentations extending into the aperture (U.S. Pat. No. 7,391,021 B2 by Stoermer et al.: “Ion guides with RF diaphragm stacks”).
In 1998, McLafferty et. al. presented a technique for fragmenting protonated proteins (Journal of American Chemical Society, 1998, vol. 120, issue 12, p. 3265-3266: “Electron capture dissociation of multiply charged protein cations. A nonergodic process”). The protonated proteins are fragmented by interacting with thermal electrons (ECD, Electron Capture Dissociation) while both are stored inside an ICR cell of a Fourier transform mass spectrometer. The magnetic field used in ICR mass spectrometers is important for ECD in these instruments. The same magnetic field that confines ions in and ICR cell is also used to radially confine electrons during the ECD process. The primary difficulty with implementing ECD on other types of mass spectrometers—i.e. instruments that do not use magnetic fields—is that the inhomogeneous RF fields of RF traps and RF ion guides, which are conventionally used to confine ions, do not confine electrons. This is because the mass of the electron is much smaller compared to the mass of ions. Electrons injected into these devices also fail to remain at near thermal energies for a time interval that is sufficient to allow ECD reactions.
In 2004, Hunt et al. presented a technique for fragmenting protonated proteins based on ion-ion reactions of protonated proteins with singly charged negative reagent ions (U.S. Pat. No. 7,534,622 B2 by Hunt et al.: “Electron transfer dissociation for biopolymer sequence mass spectrometric analysis”). Suitable negative reagent ions for electron transfer dissociation (ETD) are typically radical anions of polyaromatic compounds, such as those of fluoranthene, fluorenone and anthracene. Alternatively, it is also known that some monoaromatic or even non-aromatic compounds, such as 1-3-5-7-Cyclooctatetraen, are also suitable. The ETD reagent anions easily donate an electron to a protonated protein forming stable, neutral molecules with complete electron configuration.
The ETD reagent anions are typically generated in NCI ion sources (NCI=negative chemical ionization) by electron capture or by electron transfer. NCI ion sources have essentially the same design as chemical ionization (CI) ion sources, but they are operated in a different way in order to obtain large quantities of low-energy electrons. However, ETD reagent anions can also be generated directly or indirectly in other atmospheric pressure ionization sources, such as electrospray ionization, atmospheric pressure chemical ionization, atmospheric sampling glow discharge ionization, or other discharge sources; essentially any source that can generate an excess of thermal electrons. “Indirect generation” means that anions of selected substances are generated and subsequently converted by, for example, collision induced dissociation or metastable dissociation into radical anions that are suitable as ETD reagent anions (Huang et al., Analytical Chemistry, 2006, vol. 78, p. 7387-7391: “Electron-Transfer Reagent Anion Formation via Electrospray Ionization and Collision-Induced Dissociation”).
In U.S. Pat. No. 7,534,622 B2, Hunt et al. disclose that ion-ion reactions involving the transfer (abstraction) of electrons from multiply charged protein anions can be used to effect negative electron transfer dissociation (NETD) of the protein anions. The reagent ions suitable for NETD are singly charged radical gas-phase cations, having a polarity opposite to the protein anions.
In U.S. Pat. No. 7,534,622 B2, Hunt et al. disclose that ETD and NETD can take place inside RF containment devices, like 3D ion traps or linear ion traps with pseudo-potential barriers at their ends, or inside RF ion guides. The RF containment devices confine ions of both polarities by appropriate pseudo-potentials in all directions. RF ion guides confine ions only in the radial direction and are typically used to transfer ions in the vacuum systems of mass spectrometers, e.g. from an ion source to a mass analyzer. RF ion guides are typically designed as multipole rod systems (without axial confinement) or as RF ion funnels or tunnels formed by a stack of ring electrodes arranged along an axis.
Wu et al. demonstrated that ETD can be implemented in a linear quadrupole ion trap without mutual confinement of ions of opposite polarity (Wu et al., Analytical Chemistry, 2004, vol. 76, p. 5006-5015: “Positive Ion Transmission Mode Ion/Ion Reactions in a Hybrid Linear Ion Trap”). They describe three ways how reactions between ions of opposite polarities can be effected in “transmission modes” of a linear ion trap, whereby ions of both polarities are introduced in the axial direction. The first transmission mode involves the storage of neither ion polarity and relies on reactions taking place between the ions of opposite polarity as they are continuously admitted through the linear ion trap utilized as a RF ion guide. The second and third transmission modes involve storing ions of one ion polarity by appropriate DC potentials applied to containment lenses at the ends of the linear ion trap, whereas ions of the other polarity are continuously passing through the linear ion trap. The quadrupole field of the ion trap focuses ions of both polarities in the radial direction onto the central axis and leads to a spatial overlap of positive and negative ions and the resulting ETD reactions.
A particular application of ETD is the sequence analysis of peptides and proteins by mass spectrometry. The terms “polypeptide”, “peptide”, “oligopeptide” and “protein” refer to a polymer of amino acids without regard to the length of the polymer; thus, the terms are used interchangeably. These terms also do not specify or exclude chemical or post-expression modifications of the polypeptides. ETD promotes efficient fragmentation of peptide bonds all-over the protein backbone of proteins and thus makes it possible to deduce their amino acid sequences. The sequence analysis typically comprises the steps of: (a) generating and isolating multi-charged protein cations; (b) confining the protein cations in an RF containment device; (c) injecting ETD reagent anions into the RF containment device to facilitate electron transfer from the ETD reagent anions to the multi-charged protein cations, thus inducing the production of ETD fragment ions; and (d) acquiring a mass spectrum of the ETD fragment ions in a mass analyzer. The fragment ion spectrum contains signals arranged like ladders, and the mass distances between the signals allow to determine the amino acids and thus to deduce the amino acid sequence.